Flow-through electroporation system for ex vivo gene therapy

ABSTRACT

A method and apparatus for introducing a preselected molecule into a living cell by contacting the cell with the preselected molecule and applying a multiple series of electrical pulses to the cell. The method can be utilized ex vivo. The multiple electrical pulses generate rotating electric fields which introduce transient pores in the living cell without killing the cell. The rotating electric fields are provided in a flow through chamber apparatus having more than two electrodes. A three-step pulse process, e.g. collection, electroporation, electrophoresis, is used to introduce the preselected molecule into the cell. A mechanical means of repositioning cells between successive pulses is also provided. The apparatus can also provide a means to pulse cells at different temperatures and then after pulsing, let the cells recover for a specified residence time at another temperature.

This application is a divisional of U.S. application Ser. No.09/090,471, filed Jun. 3, 1998, now U.S. Pat. No. 6,027,488, the entirecontents of which is hereby incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates generally to the use of electric pulses toincrease the permeability of a cell and more specifically to aflow-through electroporation system.

BACKGROUND OF THE INVENTION

Electric fields can be used to create pores in cells without causingpermanent damage. This allows for insertion of large molecules into cellcytoplasm. Genes and other molecules such as pharmacological compoundscan be incorporated into live cells through a process known aselectroporation. The genes or other molecules are mixed with the livecells in a buffer medium. Short pulses of high electric fields areapplied to make the cell membranes transiently porous so that the genesor molecules can enter the cells and modify the genome of the cells.

Studies have shown that large size nucleotide sequences (e.g., up to 630kb) can be introduced into mammalian cells via electroporation (Eanault,et al., Gene (Amsterdam), 144(2):205, 1994; Nucleic Acids Research,15(3):1311, 1987; Knutson, et al., Anal. Biochem., 164:44, 1987; Gibson,et al., EMBO J., 6(8):2457, 1987; Dower, et al., Genetic Engineering,12:275, 1990; Mozo, et al., Plant Molecular Biology, 16:917, 1991).However, the efficiency of electroporation, as reflected in the currentliterature, is usually low (see U.S. Pat. No. 5,019,034, hereinincorporated by reference). A typical result is from about 5 to 20percent transfection depending on conditions, parameters and cell type.Creation of a high efficiency method and apparatus for the of transferof nucleic acid and the introduction of other preselected molecules intoliving cells via electroporation is desired.

Genetronics, Inc, San Diego, Calif., has provided an ex vivo flowthrough electroporation method and chamber in U.S. Pat. Nos. 5,676,646and 5,545,130, the disclosures of which are incorporated herein byreference.

SUMMARY OF THE INVENTION

The present invention provides a method and an apparatus for introducingpreselected molecules into a living cell by contacting the cell with thepreselected molecules and electrically applying a multiple series ofthree-step pulses to the cell. The method can be utilized ex vivo.

A three-step pulse process having steps of collection, electroporation,electrophoresis can be used to introduce preselected molecules into thecell. Each three-step pulse includes three discrete electrical impulseseach having a specified duration and strength to achieve its respectivefunction.

Each three-step pulse generates an electrical field with a particularfield orientation within a flow through chamber apparatus. A rotatingelectric field can be generated by applying multiple three-step pulseswith each three-step pulse having an electric field in a differentorientation. This rotating electric field can be configured to introducetransient pores in the living cell without killing the cell. Therotating electric field is provided in a flow through chamber apparatushaving more than two electrodes.

A mechanical means of repositioning cells between successive pulses isalso provided, e.g., a vibrating table for agitating the cell-moleculemixture. This can increase the areas of permeabiliztion of the livingcells.

An apparatus in accordance with the invention can also provide a meansto pulse cells at different temperatures and then after pulsing, let thecells recover for a specified residence time at another temperature.

The use of such features provides high viability of cells afterelectroporation and high transformation efficiency.

These and other aspects and advantages of the invention will become moreapparent in light of the following drawings, detailed description andthe appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is schematic showing a sectional view through a coaxial flowthrough chamber for generating rotating electric fields.

FIG. 2A is a schematic showing an end view of a square housing utilizedin the flow through chamber illustrated in FIG. 1.

FIG. 2B is a diagram illustrating four possible electric fieldconfigurations generated by a flow through chamber having the squarehousing illustrated in FIG. 2A.

FIG. 2C is a diagram showing an end view of a hexagonal housing utilizedin the flow through chamber illustrated in FIG. 1.

FIG. 2D is a diagram illustrating six possible electric fieldconfigurations generated by a flow through chamber having the hexagonalhousing illustrated in FIG. 2C.

FIG. 3A is an illustration of a cell in an electric field E undergoing athree step process, e.g. collection, electroporation, andelectrophoresis, for introducing pre-selected molecules into the cell.

FIG. 3B shows relative electric pulse amplitude and duration for thethree step process illustrated in FIG. 3A.

FIGS. 3C-3F illustrate several three-step pulse waveforms.

FIG. 4 illustrates a preferred embodiment of an apparatus forelectroporation mediated, ex vivo, intra cellular drug and genedelivery.

FIG. 5 is a perspective view of an alternate embodiment of pump and flowthrough chamber component having variable temperature baths.

FIG. 6 is a detail schematic of the embodiment illustrated in FIG. 5during operation.

FIG. 7 is a perspective view of another alternate embodiment of a pumpand flow through chamber mounted on a vibrating table.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIG. 1 shows a sectional view through a flow through chamber 100 forgenerating rotating electric fields according to one embodiment of theinvention. A housing 120 is provided, having walls 130 to define anelongated internal chamber 140 which extends the length 125 of thehousing 120. An inlet 110 and an outlet 115 are connected to theinternal chamber 140 to provide a conduit for continuous or pulsatingfluid flow along the length 125 of the internal chamber 140.

More than two electrodes 150 are positioned within the housing 120 andin contact with the internal chamber 140. Each of the electrodes 150 hasa section 155 that extends outside the housing 120. This section 155 ofthe electrode 150 is preferably inserted into a printed circuit boardtemplate 160 and held in place by detachable electrical contacts 170such as sliding contacts as shown. The sliding contacts 170 provide easyremoval and insertion of the flow through chamber 100. The flow throughchamber 100 can be disposable or removed from the circuit board template160 for sterilization. The circuit board template 160 is supported by asupport structure 180. The electrodes 150 are thus electricallyconnected to the circuitry in the circuit board template 160. Thecircuit board template 160 is electrically connected to an electricalsignal generating source 190.

More than two electrodes 150 are incorporated into the housing of theflow through chamber 100 in order to generate rotating electric fieldsin the internal chamber 140. Applying rotating electric fields to livingcells for electroporation can effectively increase preselected moleculeuptake and efficacy. Certain aspects of using rotating electric fieldsto enhance electroporation are described in U.S. Pat. No. 5,702,359,which is incorporated herein by reference. The electrodes 150 are formedof electrically conducting materials and may be gold plated. In oneimplementation, the electrodes 150 can be linear rods spaced apart anddisposed at the place where one wall 131 meets an adjacent wall 132 ofthe housing 120 along the length 125 of the housing 120 as shown inFIGS. 2A and 2C. The shape and the number of walls of the housing can bemodified to accommodate various numbers of linear rod electrodes. Acircular cylindrical housing, for example, is anticipated. Twoparticular configurations of the housing 120 are illustrated in FIGS. 2Aand 2C.

FIG. 2A shows an end view of a square housing utilized in a flow throughchamber illustrated in FIG. 1. The housing 120 has four walls 131,132,133, 134 which define a square elongated internal chamber 140. Thefour linear electrodes 151, 152, 153, 154 are spaced apart andpositioned within the housing 120. Each electrode 151 is positionedalong the length of an intersection between one wall 131 and an adjacentwall 132.

The four electrodes 151, 152, 153, 154 in the square housing 120 areeach connected to the electrical signal generating source 190 via theprinted circuit board template 160. The electrical signal generatingsource 190 provides pulsed electrical fields to two opposing pairs ofelectrodes so that electrical fields are respectively establishedbetween electrodes 151, 152, 153, and 154. Each pulse can have athree-step wave form, called a three-step pulse illustrated in FIGS.3A-3B. The electric field can be rotated between each successivethree-step pulse, however, the electric field is not rotated in betweeneach impulse step of the three-step pulse. After one or several pulses,a pulse generator control 197 in the electrical signal generating source190 connects another pair of electrodes in the square housing 120 whichare positioned 90 degrees from the first set up and pulses again. Thisenables each successive electric pulse to generate a different electricfield configuration which ultimately produces a rotating electric fieldwithin the elongated internal chamber 140.

The four electrodes are capable of generating four different electricfield configurations as shown in FIG. 2B. In the first configuration,electrodes 151 and 152 are connected together and electrodes 153 and 154are connected together. In the second configuration, like the firstconfiguration, the same pairs of electrodes are connected together butthe opposite charge is imposed on the electrodes. In the thirdconfiguration, electrodes 151 and 154 are connected together andelectrodes 152 and 153 are connected together. In the fourthconfiguration, like the third configuration, the same pairs ofelectrodes are connected together but the opposite charge is imposed onthe electrodes. Changing the connections of the electrodes changes theconfiguration and orientation of the electrical field and generates arotating electric field. The pulse generator control 197 can beprogramed to rotate the electric field automatically or alternatively,the pulse generator control 197 can be operated manually.

FIG. 2C is an end view of a hexagonal housing utilized in a flow throughchamber illustrated in FIG. 1. The housing 120 has six walls 131, 132,133, 134, 135, 136 which define a hexagonal elongated internal chamber140. The six linear electrodes 151, 152, 153, 154. 155, 156 are spacedapart and positioned within the housing 120. The six electrodes arecapable of generating six different electric field configurations asshown in FIG. 2D.

To introduce preselected molecules into living cells, a liquid cellsample and a fluid medium having preselected molecules are combined toform a liquid cell-molecule mixture. This mixture is then feed into theinternal chamber 140 of the flow through chamber 100 via inlet 110.Inside the internal chamber 140, the cells in the mixture are exposed torotating electric fields generated by the electrodes 150.

FIGS. 3A-3B illustrate a three-step pulse process, e.g. collection,electroporation, electrophoresis, which is used to introduce preselectedmolecules into the cell. Each three-step pulse includes three discreteelectrical impulses having a specified duration and strength to achieveits respective function. FIG. 3A shows a cell 310 in an electric field320.

In the collection step, a first electric impulse 335 is applied tocollect charged preselected molecules at regions 330 a, 330 b near thecell membrane 340. Next in the electroporation step, a second electricimpulse 355 is applied to permeabilize the cell membrane 340 formingtransient pores 360. Finally in the electrophoresis step, a thirdelectric impulse 375 is applied to transport the preselected moleculesthrough the transient pores 360 into the cell 310.

FIG. 3B shows the relative amplitude and duration of each electricimpulse in a single three-step pulse. The collection impulse 335 and theelectrophoresis impulse 375 may have similar amplitudes while theamplitude of the electroporation impulse 355 can be significantly largerthan that of the other two impulses. The amplitude of theelectroporation impulse 355 is sufficient to cause transient pores 360in the cell membrane 340. Preferably, the electroporation impulse 355creates an electric field of about 100 to 10,000 Volts per centimeter.Since the collection step and the electrophoresis step does not functionto create additional membrane permeabilization, the amplitude of thecollection impulse 335 and the electrophoresis impulse 375 is much lowerthan the amplitude of the electroporation impulse 355. Preferably thecollection impulse 335 and the electrophoresis impulse 375 each createan electric field of about 10 to 1000 Volts per centimeter.

The time duration of the high amplitude impulse 355 is shorter than thatof the other two impulses 335, 375 because a long exposure of a livingcell to the high amplitude impulse 335 may damage or even kill theliving cell. Preferred time duration ranges for each pulse are asfollows: the collection impulse 335 is about 0.1 to about 1000 ms; theelectroporation impulse 355 is about 1 to about 1000 μs; theelectrophoresis impulse 375 is about 0.1 to about 1000 ms. The longpulse length of the electrophoresis pulse 375 allows the preselectedmolecules to be “loaded” into the opened pores via an electrophoreticevent and increases the number of molecules delivered to each cell. Thusa higher percentage of cells is transfected. Also, the cells can beplaced into a bath to allow longer recovery time after the three-steppulse has been applied.

Certain selected molecules may be charged with either positive ornegative polarity. For positively charged particles, for example, onlythe molecules initially located between a target cell and the positiveelectrode may migrate to the region 330 a near the cell membrane 340 andto enter the cell. For positively charged molecules that are initiallylocated between the target cell and the negative electrode, the electricfield forces those molecules to move away from the cell membrane 340.Hence, in order to improve the efficiency of electroporation, the targetcells may be repositioned to rotate with respect to the direction of theexternal electric field by agitating the cells in the flow-throughchamber with a mechanical vibrator.

Alternatively, a rotating electric field can be generated by applying amultiple series of three-step pulses, wherein each successive three-steppulse generates an electric field with a different field orientation.Changes in field orientation produces a rotating electric field. Thus,molecules on either sides of the target cells can enter the cells andthereby improve the efficiency of the electroporation.

Such rotating electric fields can also be generated without specificallyusing a three-step pulse. A pulse having only one impulse step may alsobe used. A multiple series of this pulse can also generate a rotatingelectric field if the field orientation between each successive pulse inthe series is changed.

Multiple pulses, e.g. multiple three-step pulses, can also be applied ineach of different field orientations. Changes in field orientationbetween sets of multiple pulses can also produce a rotating electricfield.

Moreover, the above rotating electric fields and the repositioning ofthe cells by a mechanical vibrator may be used in combination to improvethe efficiency of electroporation.

The flow through chamber 100 can operate under a continuous flow mode ora batch mode, e.g. stop and go. During the continuous flow mode, liquidcell-molecule mixture is continuously fed into the flow through chamberwhile the rotating electric field is applied. In an ex vivoimplementation, the time between the withdrawal of cells from a patientand reinfusion of electroporated cell may be long when cell culturing isperformed before reinfusion. During the batch mode, the flow throughchamber is first filled with a first volume of liquid cell-moleculemixture. The flow is then stopped and multiple series of three-steppulses is applied. Next, the flow through chamber is emptied andrefilled with the second volume of liquid cell-molecule mixture.

The flow through chamber 100 can be used in an ex vivo implementationapparatus system 400 as shown in FIG. 4. This apparatus system 400comprises a peristaltic pump 410, and an injection pump 412, a flowthrough chamber 100 and a signal generator 190. The apparatus 400further comprises fluid conduit segments 424, 426, 428, 430 and 432formed of a suitable tubing along with a T-shaped coupling 434 forenabling fluid flow in the directions indicated by the wide arrows inFIG. 4. The fluid conduit segment 424 directs a cell sample withdrawnfrom a patient (e.g., by using an implantable catheter) to theperistaltic pump 410. The cell sample may be directly withdrawn from thepatient or may be cultured and processed for a certain period time.However obtained, the cell sample is mixed with a fluid medium from theinjection pump 412 to form a mixture which is sent to the flow throughchamber 100 via the fluid conduit segment 430 for electroporation. Thefluid segment 432 connected to the flow through chamber 100 is used toinfuse the electroporated mixture back to the patient by, for example,using an implantable catheter. The apparatus 400 includes a pair ofelectric cables 436 and 438 for connecting the signal generator 190 andthe flow through chamber 100.

The injection pump 412 may be of the conventional type that employs asyringe 440 for holding a quantity of a fluid medium carryingpreselected macromolecules such as genes or pharmaceutical compounds.The plunger of the syringe 440 is pushed inwardly by a motor drivenpiston assembly 442. The rate of delivery of the fluid medium from thesyringe 440 through the fluid conduit segment 426 may be adjusted viacontrol dial 444 with the delivery parameters being indicated on adisplay 446.

The peristaltic pump 410 may be implemented by a conventionalperistaltic pump and has controls for adjusting the rate of pumping. Theperistaltic pump 410 actively pumps the liquid cell sample outside thepatient 422 in a circulatory fashion. The peristaltic pump 410 includesa replaceable tubing segment 448 to propel the liquid cell sampletherethrough to the T-shaped coupling 434 where the liquid cell samplemixes with the fluid medium from the injection pump 412. This fluidmedium may be a pharmaceutical compound suspended in a suitable liquidvehicle such as saline solution. Where genes are to be introduced intothe cells of the patient, the fluid medium comprises the genes suspendedin a suitable liquid medium that will support the viability of the genesuntil they are inserted into the cells of the patient. Such fluid mediaare well known to those skilled in the art.

The details of the flow through chamber 100 are illustrated in FIGS. 1,2A, and 2C. Electrical cables 436 and 438 from the signal generator 190have plugs that are removably connected to the flow through chamber 100.These cables provide an electrical connection to the electrodes 150 ofthe flow through chamber 100.

The primary function of the electrical signal generating source 190 isto generate a predetermined electric signal which, when applied to theelectrodes 150 of the flow through chamber 100, results in applyingelectric fields of a predetermined amplitude and duration to the mixtureof liquid cell sample and fluid medium flowing therethrough.

When a cell is placed in an electrical field, an electrical potential isinduced across the cell membrane. For a spherical cell, the membranepotential induced by an electrical field is:

V _(m)=1.5RE cos θ

where R is the radius of the cell, E is the strength of the externalfield and θ is the angle between the direction of the external field andthe normal vector of the membrane at a the specific site. See U.S. Pat.No. 4,822,470, which is incorporated herein by reference.

The induced electric field within the membrane can be approximatelyrepresented by:

E _(m) =V _(m) /d=1.5(R/d)E cos θ

where d is the thickness of the membrane, and by definition is smallerthan R. The electric field in the membrane exerts a strong force on themembrane, such that pores will be formed. The pores induced by theelectric field are reversible, an introduction of molecules such asnucleic acid is possible, and most of the cells can remain viable whenthe strength of the applied electric field is properly chosen.

Preferably these fields are applied in a three step manner and thepolarizations of the fields rotate in a predetermined sequence. Pairs ofelectrodes are connected together electrically and pulsed againstanother opposing pair of electrodes producing an electric field with aspecific orientation. After one or several pulses, the pulse generatorcontrol 197 connects another pair of electrodes and pulses again. Thisproduces an electric field with a different orientation from the firstelectric field produced.

Pulse generators for carrying out the method of the invention are andhave been available on the market for a number of years. One suitablesignal generator is the ELECTRO CELL MANIPULATOR Model ECM 600commercially available from BTX, a division of Genetronics, Inc., of SanDiego, Calif., U.S.A. The ECM 600 signal generator generates a pulsefrom the complete discharge of a capacitor which results in anexponentially decaying waveform. The electric signal generated by thissignal generator is characterized by a fast rise time and an exponentialtail. In the ECM 600 signal generator, the electroporation pulse lengthis set by selecting one of ten timing resistors marked R1 through R10.They are active in both High Voltage Mode (HVM) which has a capacitancefixed at about 50 microfarads and Low Voltage Mode (LVM) which has acapacitance ranging from about 25 to about 3,175 microfarads.

The application of an electrical field across the membrane of a cellinduces transient pores which are critical to the electroporationprocess. The ECM 600 signal generator provides the voltage (in kV) thattravels across the gap (in cm) between the electrodes. This potentialdifference defines what is called the electric field strength where Eequals kV/cm. Each cell has its own critical field strength for optimumelectroporation. This is due to cell size, membrane makeup andindividual characteristics of the cell wall itself. For example,mammalian cells typically require between 0.5 and 5.0 kV/cm before celldeath and/or electroporation occurs. Generally, the required fieldstrength varies inversely with the size of the cell.

The ECM 600 signal generator has a control knob that permits theadjustment of the amplitude of the set charging voltage applied to theinternal capacitors from 50 to 500 volts in LVM and from 0.05 to 2.5 kVin HVM. The maximum amplitude of the electrical signal is shown on adisplay incorporated into the ECM 600 signal generator. This devicefurther includes a plurality of push button switches for controllingpulse length, in the LVM mode, by a simultaneous combination ofresistors parallel to the output and a bank of seven selectable additivecapacitors.

The ECM 600 signal generator also includes a single automatic charge andpulse push button. This button may be depressed to initiate bothcharging of the internal capacitors to the set voltage and to deliver apulse to the outside electrodes in an automatic cycle that takes lessthan five seconds. The manual button may be sequentially pressed torepeatedly apply the predetermined electric field.

The waveform of the voltage pulse provided by the generator in the powerpack can be in various forms. For certain applications, a square wavetrain may be preferably used as shown in FIG. 3B. FIGS. 3C-3Frespectively show some other exemplary waveforms that may be used,including but not limited to, an exponentially decaying wave train, aunipolar oscillating pulse train, or a bipolar oscillating pulse train.In addition, a three-step pulse sequence may use different pulsewaveforms for different steps.

The field strength of an electric impulse is calculated by dividing thevoltage by the distance (in cm) between the electrodes. For example, ifthe voltage is 500 V between two electrode faces which are ½ cm apart,then the field strength is 500/(½) or 1000 V/cm or 1 kV/cm.

The waveform, electric field strength and pulse duration are dependentupon the exact construction of the delivery device and types of cellsused in the electroporation procedure. One of skill in the art wouldreadily be able to determine the appropriate number of pulses useful inthe method of the invention by measuring transformation efficiency andcell survival using methods well known in the art.

The electrical pulse, preferably the three-step pulse, can be appliedwhile the cells are at any temperature, generally the electrical pulsewill be applied while the cells are at a temperature from about 2° C. to39° C. Preferably, the electrical pulse is applied while the cells areat about 2° C. to 10° C. The temperature is not changed during a singlethree-step pulse. Rather, if temperature change is desired, thetemperature change is imposed after completion of one three-step pulsesequence and before the start of another three-step pulse sequence.Following the electrical pulse, the cells can be incubated for a periodof time. Preferably, the cells are incubated at a temperature of about37° C.

FIG. 5 shows an alternative embodiment of a combination peristaltic pump510 and a flow through chamber 100 with multiple temperature bathsconnected in the fluid conduit 530. The peristaltic pump 510 has a rotorassembly 515 for receiving tubing 530. A control panel 512 providesmeans for selection and control of various parameters of the pump 510,including start-stop directional pumping and rate of pumping and time.The pump 510 is also provided with a suitable display panel 514providing a visual readout of certain selected parameters or operatingconditions. One embodiment of the flow through chamber 100 isillustrated in FIGS. 1, 2A, and 2C. The flow through chamber 100provides the multiple electrical pulses which generates a rotatingelectric field. After each pulse the mixture of cells and molecules ispumped to a specified temperature bath 540. The apparatus provides ameans to pulse the mixture at varying temperatures and allows themixture to reside in a different temperature bath after the pulse isapplied.

FIG. 6 shows a diagram of one preferred process of temperature variationafter each pulsing event. A pulsing event may be one single three-steppulse or a series of three-step pulses. A first pulsing event 610 occursin a flow through chamber 100. The mixture is pumped through tubing 530into a first temperature bath 541 for the cells to recover. After asecond pulsing event, the mixture is pumped into a second temperaturebath 542 for a second recovery. After a third temperature bath 543 canbe used to provide cell culturing or other activities depending on thespecific protocol used.

The following example is intended to illustrate but not limit theinvention. While they are typical of those that might be used, otherprocedures known to those skilled in the art may alternatively be used.One sample protocol for electroporation of human red blood cells isprovided. The First pulsing event occurs at 4° C. The electric fieldstrength generated is about 2500 Volts per centimeter while the pulsingevent lasts for about 0.3 milliseconds. The cells are allowed to recoverat about 4° C. for approximately 5 minutes. A second pulsing eventoccurs at about 37 degrees Celsius. The electric field strengthgenerated is about 1875 Volts per centimeter while the pulsing eventlasts for about 0.3 milliseconds. The cells are allowed to recover atabout 37° C. for approximately 10 minutes. The electroporated cells arethen accumulated at another water bath at about 4° C.

FIG. 7 shows another embodiment where the flow through chamber 100 ismounted on a vibrating table 710. The vibrating table 710 producesvibrate the flow through chamber 100 to mechanically agitate thecell-molecule mixture between successive pulses, e.g. successivethree-step pulses. This repositions cells and can potentially increasethe areas of permeabilization to improve the electroporation efficiency.The degree and duration of the vibration can be adjusted by a controlmeans 715 which is coupled to the vibrating table 710. The degree of thevibration should be sufficient to slightly turn the cells' orientationto the electrodes.

A number of embodiments of the present invention have been described.Nevertheless, it will be understood that various modifications andenhancements may be made without departing from the spirit and scope ofthe invention as defined by the following claims.

What is claimed is:
 1. An ex vivo method for introducing pre-selectedmolecules into cells of a host, comprising: contacting ex vivo the cellswith the pre-selected molecules to form a cell-molecule mixture; placingthe cell-molecule mixture in a chamber selected from the groupconsisting of a continuous flow chamber and a batch flow chamber,wherein the chamber has an inner surface that contacts the cell-moleculemixture and on which are disposed at least four electrodes positioned inspaced apart relation to one another, wherein the electrodes form atleast two opposed pairs of electrodes such that the electrodes of agiven pair are disposed opposite one another on the inner surface of thechamber; and pulsing the cell-molecule mixture with at least oneelectric impulse between at least one of the opposed pairs to apply atleast one electric field orientation to the cell-molecule mixture,thereby causing at least a portion of the pre-selected molecules to beintroduced into at least a portion of the cells.
 2. The method of claim1, wherein the at least four electrodes are gold-plated electrodes. 3.The method of claim 1, wherein the method further comprisesadministering a series of electric impulses to apply electric fields ina changing field orientation to produce a rotating electric field. 4.The method of claim 1, wherein the at least four electrodes are sixelectrodes and the method further comprises administering a series ofelectric impulses to apply electric fields in a changing fieldorientation to produce a rotating field.
 5. The method of claim 1,further comprising obtaining the cells from a donor patient.
 6. Themethod of claim 5, further comprising reintroducing the cells containingthe selected molecules into the donor patient after culturing.
 7. Themethod of claim 5, further comprising culturing the cells obtained formthe donor patient.
 8. The method of claim 7, wherein the cells arecultured after the selected molecules are introduced into the cells. 9.The method of claim 7, further comprising reintroducing the cellscontaining the selected molecules into the donor patient afterculturing.